Blue collagenase assay

ABSTRACT

A method of measuring soluble or insoluble cell or tissue-associated collagenase activity. The substrate includes native (fibrillar) collagen fragments that were stained with Coomassie Brilliant Blue R-250. Incubation with collagenase can be observed in real-time by the generation of digested smaller fragments. The degraded blue fragments are obtained by filtration through class fibers, onto which intact collagen fibrils are retained. The filtrate containing the blue collagen fragments is incubated with a detergent in order to extract the blue dye, and the mixture is centrifuged in order to separate the dye in the supernatant from the pellet, which contains de-stained collagen fragments and other insoluble materials contained in the test samples (such as bacterial cells or tissues). The amount of dye extracted is quantified by measuring the amount of dye extracted from these fragments, i.e., the absorbance at 600 nm using a spectrophotometer or an ELISA reader.

CROSS-REFERENCE TO RELATED APPLICATIONS

This nonprovisional application is a continuation of and claims priorityto U.S. Provisional Patent Application No. 62/020,179, entitled “BlueCollagenase Assay”, filed Jul. 2, 2014, by the same inventor, theentirety of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates, generally, to collagenases. More specifically,it relates to blue collagenase assays.

2. Brief Description of the Prior Art

Collagenases are defined as proteases that can digest native collagen inthe triple helix region. There is no spectrophotometric assay that usesnative collagen fibrils [“Collagenase”, Worthington Biochemical Company,I.U.B, 3.4.24.3, C.A.S. 9001-12-1]. Commonly used substrates incollagenase assay kits include synthetic peptides, or denatured collagensuch as acid soluble collagen and heat-gelling collagen or gelatin.Since denatured collagen can also be degraded by gelatinase and/orprotease such as trypsin, assays based on the digestion of thesesubstrates are not specific for the determination of collagenaseactivity, and hence are referred to by those of ordinary skill in theart as collagenolytic activity, which can be broadly defined as just theability to lyse collagen, gelatin, and other proteins containingproline.

Collagen can be stained in blue tissue section by blue dyes such asaniline blue dyes (e.g., MSDS: CI#: 42755; Synonym: Acid blue 22 Chinablue; Chemical Name: Not available; Chemical Formula: C32H27N309S3.2Na)and Coomassie Brilliant Blue R-250 (MSDS: C14: 42660; Synonym: Acid Blue83; Acid Brilliant Blue; CI Acid Blue 83; Chemical Name:Benzenemethanaminium,N-[4-[[4-[(4-ethoxyphenyl)amino]phenyl][4-[ethyl[(3-sulfophenyl)methyl]amino]phenyl]methylene]-2,5-cyclohexadien-1-ylidene]-ethyl-3-sulfo-,inner salt, monosodium salt; Chemical Formula: C46-17144-N3-O7-S2-Na).This property has been applied to the preparation of bluecollagen-coated plastic plates (gelled collagen and dried onto plasticplates) for the observation of collagen degradation assay by oralsquamous carcinoma cells in cancer research (Aznavorian et al., CancerResearch, American Association for Cancer Research, 2001:61:6264-6275),and adapted to spectrophotometric measurement of the plates (Nethery etal., “A spectrophotometric collagenase assay”, 1986, Anal Biochem159(2):390-5). Since the collagen is denatured in the heat-gellingprocess, the use of inhibitors of other enzymes is applied in order todeduce that the activity observed is that of collagenase (Aznavorian etal., Cancer Research, American Association for Cancer Research,2001:61:6264-6275).

The interest in the detection and measurement of collagenase activityand/or use of Coomassie dye has generated the publication of numerousassays. Examples include EP 2016421 B1; U.S. Pat. No. 6,057,160; U.S.Pat. No. 4,219,337; U.S. Pat. No. 4,023,933; EP 0319334 A2; U.S. Pat.No. 4,176,009; EP 1039299 B1; Sedmak et al. A rapid, sensitive, andversatile assay for protein using Coomassie brilliant blue 0250,Analytical Biochemistry. Volume 79, Issues 1-2, May 1, 1977, pp.544-552; Burokerkilgore et al. A Coomassie Brilliant Blue 0-250-BasedColorimetric Assay for Measuring Activity of Calpain and OtherProteases. Analytical Biochemistry. Volume 208, Issue 2, Feb. 1, 1993,pp. 387-392; and Teratoa et al. A rapid assay method of collagenaseactivity using 14C-labeled soluble collagen as substrate. Biochimica etBiophysica Acta (BBA)—Enzymology. Volume 445, Issue 3, Oct. 11, 1976,pp. 753-762. However, each of these references has their own respectivedrawbacks and would be incapable, individually or in combination, ofmeasuring soluble or insoluble cell or tissue-associated collagenaseactivity using Coomassie dye.

Accordingly, what is needed is an improved assay, kit, and/or vessel fordetection of activity of blue collagenase. However, in view of the artconsidered as a whole at the time the present invention was made, it wasnot obvious to those of ordinary skill in the field of this inventionhow the shortcomings of the prior art could be overcome.

All referenced publications are incorporated herein by reference intheir entirety. Furthermore, where a definition or use of a term in areference, which is incorporated by reference herein, is inconsistent orcontrary to the definition of that term provided herein, the definitionof that term provided herein applies and the definition of that term inthe reference does not apply,

While certain aspects of conventional technologies have been discussedto facilitate disclosure of the invention, Applicants in no way disclaimthese technical aspects, and it is contemplated that the claimedinvention may encompass one or more of the conventional technicalaspects discussed herein.

The present invention may address one or more of the problems anddeficiencies of the prior art discussed above. However, it iscontemplated that the invention may prove useful in addressing otherproblems and deficiencies in a number of technical areas. Therefore, theclaimed invention should not necessarily be construed as limited toaddressing any of the particular problems or deficiencies discussedherein.

In this specification, where a document, act or item of knowledge isreferred to or discussed, this reference or discussion is not anadmission that the document, act or item of knowledge or any combinationthereof was at the priority date, publicly available, known to thepublic, part of common general knowledge, or otherwise constitutes priorart under the applicable statutory provisions; or is known to berelevant to an attempt to solve any problem with which thisspecification is concerned.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made tothe following detailed description, taken in connection with theaccompanying drawings, in which:

FIG. 1A depicts a collagen suspension incubated with trypsin,

FIG. 1B depicts a collagen suspension incubated with C. histolyticumcollagenase.

FIG. 2 depicts collagenase activity in S. mutans (Sm).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In the following detailed description of the preferred embodiments,reference is made to the accompanying drawings, which form a partthereof, and within which are shown by way of illustration specificembodiments by which the invention may be practiced. It is to beunderstood that other embodiments may be utilized and structural changesmay be made without departing from the scope of the invention.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural referents unless the contentclearly dictates otherwise. As used in this specification and theappended claims, the term “or” is generally employed in its senseincluding “and/or” unless the context clearly dictates otherwise.

Blue collagenase assay is a method of measuring soluble or insolublecell or tissue-associated collagenase activity. The substrate includesnative fibrillar collagen fragments that were stained with CoomassieBrilliant Blue R-250. Incubation with collagenase can be Observed inreal-time by the generation of digested smaller fragments, and can beobtained by filtration, and can be quantitated by measuring the amountof dye extracted from these fragments.

The present method is amenable to the preparation of a blue collagenaseassay kit, which would contain reaction tubes containing blue collagensubstrate, tips plugged. with glass wool for filtration and collectiontubes containing the extraction solution, and a bottle of concentratedsubstrate buffer.

In an embodiment, the current invention is a novel method to assay forcollagenase activity using collagen fibrils (e.g., Type I) from BovineAchilles tendon (SIGMA-ALDRICH, Product no. C9879). This method allowsfor both the qualitative observation of collagenase activity and thequantitative activity assay using a spectrophotometer. It iscontemplated and shown herein that this method is applicable to bothsoluble and cell-associated collagenase.

In certain embodiments, the method presented herein is unique instaining lyophilized collagen with Coomassie Brilliant Blue R-250(though other dyes are contemplated herein) at saturation level and inits native state without solubilization by acid and/or gelling by heat.Further, collagen fibrils can be cut with sharp scissors, and the dyecan be extracted from the digested collagen fragments by Polysorbate 20(TWEEN 20) for quantitative measurement of the absorbance at the optimalwave length of 600 nm, at which particulates also get absorbed. As such,the assay is applicable either to both soluble and insoluble cell or totissue-associated collagenase. Application of the Blue Collagenase Assayto soluble Clostridium histolyticum collagenase and to cell-associatedStreptococcus mutans collagenase is described in preparation.

In application, the present method can be applied to the staining otherinsoluble proteins, such as gelatin granules and other types of collagenfor use as a substrate for the analysis of gelatinase and othercollagenase types. The present method can also be used to analyzemetalloproteinases in humans and animals.

EXAMPLE 1

As an example, application of the blue collagenase assay to solubleClostridium histolyticum collagenase (see FIGS. 1A-IB) andcell-associated Streptococcus mutans collagenase (see FIG. 2) isdescribed herein.

The method generally includes the following steps:

1. The collagen fibrils are stained using Coomassie Brilliant Blue R-250(C46-H44-N3-07-S2-Na).

2. The Blue collagen fibrils are suspended in collagenase substratebuffer (50 mM Tris, 50 mM NaCl, 10 mM CaCl₂, pH7.5).

3. The blue collagen fibrils in suspension are incubated with the testsample at 37° C. on a rotator.

4. The collagenase activity results in the production of blue collagenparticulates that can be readily observed.

5. After the incubation time of three (3) hours or longer, the mixtureis filtered through glass wool/fibers to which the blue collagen fibrilsare retained, and the small blue fragments are collected in thefiltrate,

6, To determine the amount of digested collagen, the blue dye isextracted by incubation of the filtrate in the presence of Polysorbate20 (PATEN 20) added at 2%, at 37° C. on a rotator,

7. The mixture, containing the blue dye, is centrifuged at 12,000×g for5 minutes.

8. Absorbance of the blue supernatant is measured at 600 nm using aspectrophotometer or ELISA reader and expressed as equivalent U ofClostridium histolyticum collagenase.

For analysis, a standard curve was established with C. histolyticumcollagenase used at various concentrations: 5 U/mL (1), 10 U/mL (2), 15U/mL (3) and 20 U/mL (4) S. rautans GS-5 cell suspension was assayed inparallel using 1 mL of a bacterial suspension corresponding to 5 OD at600 nm. All samples were tested in quadruplicates. Each columnrepresents the mean of 4 measurements with standard error bar.

The reaction mixture (2 mL) consisted of 250 μl of blue collagensuspension in 1 mL Collagenase substrate buffer (CSB) and each testsample was diluted in CSB up to 1 mL. Each sample was assayed inquadruplicates. Incubation was for 4 h at 37° C. on a rotator.

The deduced activity of Sm collagenase was 9 C. histolyticum equivalentcollagenase units (9 Ch eq.U) in a sample containing S. mutans GS5 cellsat an absorbance of 5 OD at 600 nm, corresponding to a relative specificactivity of 1.8 Ch eq. U/OD of bacteria,

EXAMPLE 2 1. Staining of Collagen

Type I fibrillar Collagen from Bovine Achilles Tendon (SIGMA-ALDRICH,product no. C9879) was cut with a pair of sharp scissors into shorterfibrils. The fibrils were passed through a 1 mm sieve and collected asdry collagen.

The dry collagen was mixed with 0.2% Coomassie Brilliant Blue in asolution of 10% acetic acid, 40% methanol in deionized water (standardmethod for staining protein bands in polyacrylamide gel), at a ratio of500 mg collagen in 30 mL acetic acid-methanol-water solution, in a 50 mLconical centrifuge tube on a rotor at room temperature for 30 mins to 1hour until all the collagen was saturated with the blue dye, after whichtime acetic acid-methanol-water (10:40:50) was added up to 50 mL.

The blue collagen suspension was centrifuged at 200×g for 2 minutes, andthe supernatant was discarded. The excess dye was removed by rinsingwith the solution of 10% Acetic acid, 40% methanol in deionized water.

With repeated mixing, the blue collagen was extensively washed with PBS(phosphate buffered saline, pH 7.2.) added up to 50 mL, followed bycentrifugation and pouring off the supernatant containing floatingdenatured collagen until the supernatant came out clear and colorless.

The blue collagen was washed once with a solution of collagenasesubstrate buffer (50 mM Tris, 50 mM sodium chloride and 10 mM. Calciumchloride, pH 7.5, with sodium azide added at 0.02%) and thenre-suspended in the same, but fresh, substrate buffer.

Aliquots of the blue collagen suspension (250 μl) were distributed intograduated 2 mL microcentrifuge tubes (FISHER SCIENTIFIC Co.) using 1 mLpipettor and a cut-off pipet tip to pipet up the collagen fibrils. Thefilled microcentrifuge tubes were stored in the refrigerator until used.The blue collagen suspension is stable, even when stored at roomtemperature (25° C.). Alternatively, the blue collagen can belyophilized.

Quality control for specificity can be performed by incubation withTrypsin.

2. Establishment of a Standard Curve Using Soluble C. HistolytieumCollagenase of Known Enzyme Activity

C. hislolyticum collagenase with known enzyme activity (SIGNMA ALDRICH)and the samples to be tested were analyzed in parallel. C. histolyticumcollagenase was selected as a test sample for the establishment of astandard curve. C. histolyticum collagenase Type I of known enzymeactivity (SIGMA-ALDRICH) was solubilized in substrate buffer at 1U perμL. A standard curve is established with the C. histoyticum collagenase,which is used as a reference. Depending on the amount of enzyme in thetest samples a standard curve can be established with C. histolyticumenzyme concentrations from 20-300 Units/mL. A quick test can beperformed with C. histolyticum and the test samples in order todetermine the appropriate range of the concentrations needed for thestandard curve. This is facilitated by the observation of collagendegradation in real time.

Up to 1.75 mL of sample can be used and added to each tube containingthe blue collagen (total volume of the reaction mix is 2 mL).

The tubes were incubated at 37° C. on a rotator that are placed in anincubator for 3 hours or as long as needed depending on enzymeconcentration. Digestion of the blue collagen by collagenase wasperiodically observed, resulting in a dose-dependent generation of smallblue collagen particles. At the end of the incubation period, thereaction mixture from each tube was filtered through loosely packedglass wool.

After the incubation period, the reaction mixture was poured into 1 mLpipet tip containing loosely packed glass fibers, and the filtratecontaining small blue collagen fragments were collected into a fresh 2mL-microcentrifuge tube containing 200 μl of 20% Tween 20 in substratebuffer was added.

The tubes were then incubated at 37° C. on a rotator for about 2 hoursuntil dye was extracted from the collagen particles.

After the extraction period, the tubes were centrifuged at 10,000×g for5 minutes to sediment the de-stained collagen fragments and otherinsoluble components in the test samples. The supernatant containing thedye from each tube is measured for the absorbance at 600 nm using aspectrophotometer or an ELISA reader. Tubes incubated with buffer alone(1 mL substrate buffer containing 2% Polysorbate 20 (TWEEN 20)) wereused as a blank.

A standard curve is established with the C. histolyticum collagenase(U/mL versus OD at 600 nm), and the corresponding enzyme activity of thetest sample is derived from the curve and expressed in Ch-equivalent U,while the relative specific activity is calculated as follows:Ch-equivalent U/mg of protein (if known), or in the case of bacterialsuspension (OD 600 mn of bacteria).

As a side note, due to the heterogeneity of the collagen fibrils, astandard curve typically is established for each lot of blue collagen.

3. Activity Assay of Bacterial Cell-Associated Collagenase

Streptococcus mutans whole cells were used as a model of insolublecollagenase.

Bacterial cells were collected by centrifugation and the cell wassuspended in substrate buffer at a concentration of 5 OD_(600 nm) permL.

One (1) mL of bacterial cell suspension was added to each blue collagensubstrate tube, and substrate buffer was added up to 2 mL. The assay wasperformed in quadruplicate.

Subsequent steps were performed, as described previously for C.histolyticum collagenase.

Enzyme activity unit was derived from the C. histolyticum standard curveand expressed as C. histolyticum equivalent units.

The advantages set forth above, and those made apparent from theforegoing description, are efficiently attained. Since certain changesmay be made in the above construction without departing from the scopeof the invention, it is intended that all matters contained in theforegoing description or shown in the accompanying drawings shall beinterpreted as illustrative and not in a limiting sense.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed, and all statements of the scope of the invention that, as amatter of language, might be said to fall therebetween.

What is claimed is:
 1. An assay for measuring soluble or insoluble cellor tissue-associated collagenase activity, comprising: obtaining drycollagen fibrils suitable for dying; staining said collagen fibrils withblue dye at saturation level; suspending said blue collagen fibrils in acollagenase substrate buffer; incubating said blue collagen fibrilsuspension with a test sample at 37° C. on a rotator, whereincollagenase activity results in production of blue collagen particulatesthat can be readily observed in a resulting mixture; filtering saidmixture to retain said blue collagen fibrils, wherein said hlu collagenparticulates are digested and collected in a resulting filtrate;extracting said blue dye from said filtrate to determine an amount ofsaid digested blue collagen particulates; and measuring absorbance ofsaid digested blue collagen particulates.
 2. An assay as in claim 1,wherein the step of measuring absorbance is performed using aspectrophotometer at an optimal wave length of 600 nm.
 3. An assay as inclaim 1, wherein the step of staining said collagen fibrils is performedusing 0.2% Coomassie Brilliant Blue in a solution of aceticacid-methanol-water (10:40:50, v:v:v), at a ratio of 500 mg collagen in30 mL acetic acid-methanol-water solution.